Compositions and methods for identifying agents which modulate PTEN function and PI-3 kinase pathways

Abstract

Methods are provided for the identification, biochemical characterization and therapeutic use of agents which impact PTEN, p53, PI-kinase and AKT mediated cellular signaling.

What is claimed is:  
     
         1 . A method for identifying agents which modulate PTEN activity, comprising: 
 a) providing a host cell wherein PTEN is expressed;    b) contacting said host cell with a test agent suspected of modulating PTEN activity;    c) assessing said modulation as a function of alterations in activated AKT levels in the presence of said agent.    
     
     
         2 . A method as claimed in  claim 1 , wherein said PTEN is mutated.  
     
     
         3 . A method as claimed in  claim 2 , wherein said mutation in PTEN is selected from the group consisting of G129E, G129R, R130M and C124S.  
     
     
         4 . A method as claimed in  claim 2 , wherein said PTEN is inactive and said test agent suppresses concomittant AKT activating activity.  
     
     
         5 . A test agent identified by the method of  claim 4 .  
     
     
         6 . A method for gene therapy for the treatment of cancers arising from a mutation in PTEN, comprising administration of a nucleic acid encoding wild type PTEN to a patient in need thereof.  
     
     
         7 . The method as claimed in  claim 6 , wherein said PTEN encoding nucleic acid is inserted into a vector having tropism for said cancer cell.  
     
     
         8 . The method as claimed in  claim 7 , wherein said vector is selected from the group consisting of retroviral vectors, adenoviral vectors, shuttle vectors, disabled vaccinia viral vectors, and plasmid vectors.  
     
     
         9 . The method as claimed in  claim 8 , wherein said plasmid vector is encased in an antibody studded liposome, said antibody being immunologically specific for an antigen present on a tumor cell.  
     
     
         10 . The method as claimed in  claim 9 , wherein said tumor cell is a glioma cell and said antigen is the epidermal growth factor receptor.  
     
     
         11 . A method as claimed in  claim 10 , wherein said liposome is a cationic liposome.  
     
     
         12 . A method as claimed in  claim 7 , wherein said vector disposed in a biologically compatible medium is microinjected directly into said cancer cell.  
     
     
         13 . A method for identifying agents which modulate PTEN angiogenic activity, comprising: 
 a) providing a host cell wherein PTEN is expressed;    b) contacting said host cell with a test agent suspected of modulating PTEN angiogenic activity;    c) assessing said modulation as a function of alterations in microvessel density formation in the presence of said agent.    
     
     
         14 . A method as claimed in  claim 13 , wherein said PTEN is mutated.  
     
     
         15 . A method as claimed in  claim 14 , wherein said mutation in PTEN is selected from the group consisting of G129E, G129R, R130M and C124S.  
     
     
         16 . A method as claimed in  claim 13 , wherein said PTEN is inactive and said test agent inhibits PTEN mediated angiogenic activity.  
     
     
         17 . A method as claimed in  claim 13 , wherein said microvessel density formation determined via immunospecific binding of anti-CD31 antibodies.  
     
     
         18 . A test agent identified by the method of  claim 16 .  
     
     
         19 . A method for identifying agents which modulate PTEN activity, comprising: 
 a) providing a host cell wherein PTEN is expressed;    b) contacting said host cell with a test agent suspected of modulating PTEN activity;    c) assessing said modulation as a function of alterations in upregulation of TSP-1 in the presence of said agent.    
     
     
         20 . A method as claimed in  claim 19 , wherein said PTEN is mutated.  
     
     
         21 . A method as claimed in  claim 20 , wherein said mutation in PTEN is selected from the group consisting of G129E, G129R, R130M and C124S.  
     
     
         22 . A method as claimed in  claim 20 , wherein said PTEN is inactive and said test agent restores PTEN mediated upregulation of TSP-1.  
     
     
         23 . A test agent identified by the method of  claim 22 .  
     
     
         24 . A method for identifying agents which modulate PTEN activity, comprising: 
 a) providing a host cell wherein PTEN is expressed;    b) contacting said host cell with a test agent suspected of modulating PTEN activity;    c) assessing said modulation as a function of alterations in VEGF levels in the presence of said agent.    
     
     
         25 . A method as claimed in  claim 24 , wherein said PTEN is mutated.  
     
     
         26 . A method as claimed in  claim 25 , wherein said mutation in PTEN is selected from the group consisting of G129E, G129R, R130M and C124S.  
     
     
         27 . A method as claimed in  claim 26 , wherein said PTEN is inactive and said test agent restores regulated PTEN inhibition of VEGF production.  
     
     
         28 . A test agent identified by the method of  claim 27 .  
     
     
         29 . A method for identifying agents which modulate PTEN activity, comprising: 
 a) providing a host cell wherein PTEN is expressed;    b) contacting said host cell with a test agent suspected of modulating PTEN activity;    c) assessing said modulation as a function of alterations in TIMP3 levels in the presence of said agent.    
     
     
         30 . A method as claimed in  claim 29 , wherein said PTEN is mutated.  
     
     
         31 . A method as claimed in  claim 30 , wherein said mutation in PTEN is selected from the group consisting of G129E, G129R, R130M and C124S.  
     
     
         32 . A method as claimed in  claim 30 , wherein said PTEN is inactive and said test agent restores PTEN mediated induction of TIMP3.  
     
     
         33 . A test agent identified by the method of  claim 32 .  
     
     
         34 . A method for identifying agents which modulate PTEN activity, comprising: 
 a) providing a host cell wherein PTEN is expressed;    b) contacting said host cell with a test agent suspected of modulating PTEN activity;    c) assessing said modulation as a function of alterations in MMP-9 levels in the presence of said agent.    
     
     
         35 . A method as claimed in  claim 36 , wherein said PTEN is mutated.  
     
     
         36 . A method as claimed in  claim 35 , wherein said mutation in PTEN is selected from the group consisting of G129E, G129R, R130M and C124S.  
     
     
         37 . A method as claimed in  claim 36 , wherein said PTEN is inactive and said test agent restores regulated PTEN suppression of MMP9 activity.  
     
     
         38 . A test agent identified by the method of  claim 37 .  
     
     
         39 . A method for identifying agents which modulate PTEN activity, comprising: 
 a) providing a host cell wherein PTEN is expressed;    b) contacting said host cell with a test agent suspected of modulating PTEN activity;    c) assessing said modulation as a function of alterations of invasiveness of said cells in the presence of said agent.    
     
     
         40 . A method as claimed in  claim 39 , wherein said PTEN is mutated.  
     
     
         41 . A method as claimed in  claim 39 , wherein said mutation in PTEN is selected from the group consisting of G129E, G129R, R130M and C124S.  
     
     
         42 . A method as claimed in  claim 40 , wherein said PTEN is inactive and said test agent restores PTEN mediated inhibition of invasiveness.  
     
     
         43 . A test agent identified by the method of  claim 42 .  
     
     
         44 . A method for identifying an agent which modulates PTEN phosphatase activity comprising: 
 a) providing an enzymatically active PTEN molecule or peptide fragment in a biological buffer;    b) adding to said buffer a substrate of said PTEN enzyme, said enzymatic action of PTEN on said substrate giving rise to a detectable reaction product;    c) contacting said active PTEN molecule or fragment in said biological buffer with an agent suspected of modulating said PTEN phosphatase activity;    d) measuring said PTEN phosphatase activity in the presence and absence of said agent.    
     
     
         45 . A method as claimed in  claim 44 , wherein said PTEN is immobilized on a solid support.  
     
     
         46 . A method as claimed in  claim 45 , wherein said PTEN activity is assessed in a high throughput format.  
     
     
         47 . A method as claimed in  claim 44 , wherein said PTEN activity is contacted with a plurality of test agents.  
     
     
         48 . A method as claimed in  claim 44 , wherein a Malachite green assay is performed to determine said enzymatic activity of PTEN in the presence and absence of said test agent.  
     
     
         49 . A method for identifying agents having binding affinity for PTEN or peptide fragment thereof, said method comprising: 
 a) providing a PTEN molecule or peptide fragment in a biological buffer;    b) contacting said PTEN molecule or fragment in said biological buffer with a detectably labeled agent suspected of having binding affinity for said PTEN or peptide fragment thereof, such that a detectably labeled complex forms between those agents having affinity for said PTEN or fragment thereof; and    d) identifying and isolating said detectably labeled complex if present, thereby identifying said agent.    
     
     
         50 . A method as claimed in  claim 49 , wherein said PTEN or PTEN peptide fragment is adsorbed to a solid support.  
     
     
         51 . A method as claimed in  claim 50 , wherein said method is performed in a high throughput screening format.  
     
     
         52 . A method as claimed in  claim 49 , wherein said PTEN or fragment thereof is contacted with a plurality of detectably labeled agents present in a chemical combinatorial library.  
     
     
         53 . A method as claimed in  claim 49 , wherein said PTEN or PTEN fragment is expressed on the surface of a phage, and said expressed PTEN or PTEN fragment is contacted with a plurality of detectably labeled agents present in a chemical combinatorial library.  
     
     
         54 . A method as claimed in  claim 49 , wherein said PTEN fragment consists essentially of a fragment selected from the group consisting of DLDLTYIYP (SEQ ID NO: 3), YLVLTL (SEQ ID NO: 6), YRNNIDD (SEQ ID NO: 8), KGVTIPSQRRYVYYYSYLL (SEQ ID NO: 15), YSYL (SEQ ID NO: 7), YFSPN (SEQ ID NO: 5), RYSDTTDS (SEQ ID NO: 16), HCKAGKR (SEQ ID NO: 9), DHNPPQ (SEQ ID NO: 10), KGVTIPSQRRY SEQ ID NO: 17), HFWVNTFFI (SEQ ID NO: 11), TLTKNDLD-FFTKTV (SEQ ID NO: 12), GDIKVEF-FTKTV (SEQ ID NO: 13), DKANKDKAN-FTKTV (SEQ ID NO: 14), and HTQITKV (SEQ ID NO: 18).  
     
     
         55 . A method as claimed in  claim 53 , wherein said phage express a PTEN fragment consisting essentially of a fragment selected from the group consisting of DLDLTYIYP (SEQ ID NO: 3), YLVLTL (SEQ ID NO: 6), YRNNIDD (SEQ ID NO: 8), KGVTIPSQRRYVYYYSYLL (SEQ ID NO: 15), YSYL (SEQ ID NO: 7), YFSPN (SEQ ID NO: 5), RYSDTTDS (SEQ ID NO: 16), HCKAGKR (SEQ ID NO: 9), DHNPPQ (SEQ ID NO: 10), KGVTIPSQRRY (SEQ ID NO: 15), HFWVNTFFI (SEQ ID NO: 11), TLTKNDLD-FTKTV (SEQ ID NO: 12), GDIKVEF-FTKTV (SEQ ID NO: 13), DKANKDKAN-FTKTV SEQ ID NO: 14), and HTQITKV (SEQ ID NO: 18).  
     
     
         56 . A method for preventing or inhibiting inflammatory disease in a patient in need thereof, comprising the administration of an effective amount of a PTEN agonist.  
     
     
         57 . A method as claimed in  claim 56 , wherein said inflammatory disease is selected from the group consisting of macular degeneration, arthritis, asthma, hay fever, systemic lupus erythrematosis, Crohn's disease, and inflammatory bowel disease.  
     
     
         58 . A method as claimed in  claim 56 , further comprising the administration of an inhibitor of PI-kinase.  
     
     
         59 . A method as claimed in  claim 58 , wherein said PI-kinase inhibitor is LY294002.  
     
     
         60 . A method as claimed in  claim 56 , further comprising the administration of an AKT inhibitor.  
     
     
         61 . A method for the treatment of cancer in a patient in need thereof, comprising the administration of an effective amount of a PTEN agonist.  
     
     
         62 . A method as claimed in  claim 61 , wherein said PTEN agonist effectively blocks cancer cell metastasis.  
     
     
         63 . A method as claimed in  claim 61 , wherein said PTEN agonist effectively blocks angiogenesis.  
     
     
         64 . A method as claimed in  claim 61 , further comprising the administration of at least one additional chemotherapeutic agent.  
     
     
         65 . A method as claimed in  claim 64 , wherein said at least one additional chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, asparaginase, vincristine, vinblastine, anthracyclines, microtubule disrupting agents, taxol, herceptin, and etoposides.  
     
     
         66 . A method as claimed in  claim 61 , further comprising the administration of an inhibitor of PI3 kinase.  
     
     
         67 . A method as claimed in  claim 66 , wherein said PI-kinase inhibitor is LY294002.  
     
     
         68 . A method as claimed in  claim 61 , further comprising the administration of an inhibitor of AKT.  
     
     
         69 . A method for inhibiting p53 mediated programmed cell death in a patient in need thereof, said method comprising the administration of a PTEN inhibitor.  
     
     
         70 . A method for enhancing the chemosensitivity of tumor cells in a patient in need thereof, said method comprising the administration of an PTEN agonist to a patient having a chemoresistant tumor.  
     
     
         71 . A method for enhancing the radiosensitivity of a tumor cells in a patient in need thereof, said method comprising the administration of a PTEN agonist to a patient having a radioresistant tumor.  
     
     
         72 . A method of gene therapy for the treatment of an inflammatory condition in a patient having a mutation in PTEN, said method comprising delivery of a native PTEN encoding nucleic acid to immune cells of said patient.  
     
     
         73 . A method as claimed in  claim 72 , further comprising the administration of a PI3 kinase inhibitor.  
     
     
         74 . A method as claimed in  claim 72 , wherein said immune cell is selected from the group consisting of mast cells, B cells, T cells, dendritic cells, neutrophils, eosinophils and macrophages.  
     
     
         75 . A method for inhibiting immunoreceptor signaling in a patient in need thereof, comprising administration of an effective amount of a PTEN agonist.  
     
     
         76 . A method as claimed in  claim 75 , wherein said immunoreceptor is selected from the group consisting of a T cell receptor, B cell receptor, ITAM-bearing receptor, FcγR, FcεcR, and FcαR.  
     
     
         77 . A method as claimed in  claim 75 , wherein said agonist is administered to prevent a condition selected from the group consisting of graft rejection and graft versus host disease.  
     
     
         78 . A method for augmenting an immune reaction in a patient in need thereof, comprising administration of an effective amount of an inhibitor of PTEN.  
     
     
         79 . A method as claimed in  claim 78 , wherein said inhibitor is targeted to a cell selected from the group consisting of T cells, B cells, and macrophages.  
     
     
         80 . A method for inhibiting aberrant angiogenesis in a patient in need thereof, said method comprising the administration of a PI3 kinase inhibitor.  
     
     
         81 . A method as claimed in  claim 80 , wherein said aberrant angiogenesis is caused by cancer, autoimmune disease, arthritis, systemic lupus erthymatosis, inflammatory bowel disease, coronary artery disease, cerebrovascular disease, and atherosclerosis.  
     
     
         82 . A method as claimed in  claim 80 , further comprising the administration of an AKT inhibitor.  
     
     
         83 . A method for inhibiting aberrant angiogenesis in a patient in need thereof, said method comprising the administration of an AKT inhibitor.  
     
     
         84 . A method as claimed in  claim 83 , wherein said aberrant angiogenesis is caused by cancer, autoimmune disease, arthritis, systemic lupus erthymatosis, inflammatory bowel disease, coronary artery disease, cerebrovascular disease, and atherosclerosis.  
     
     
         85 . A method as claimed in  claim 83 , further comprising the administration of an PI3 kinase inhibitor.  
     
     
         86 . A method for inhibiting p53 mediated programmed cell death in a patient in need thereof, comprising the targeted administration of a PTEN inhibitor to normal tissues to inhibit stress induced apoptosis thereof, wherein said patient is in need for such treatment due to a condition selected from the group consisting of myocardial infarction, cerebrovascular insult and gram negative sepsis.  
     
     
         87 . A method for inhibiting p53 mediated programmed cell death in a patient in need thereof, comprising the targeted administration of a PTEN inhibitor, said PTEN inhibitor inhibiting cellular senescence thereby promoting survival of normal cells.  
     
     
         88 . A method as claimed in  claim 87 , wherein said normal cells are selected from the group consisting of brain cells, heart cells, and skin cells.